A method for stabilizing prostate specific antigen standard, a matrix calibration method and a detection kit evaluation method

Abstract

This invention discloses a method for stabilizing prostate-specific antigen (PSA) standards, a matrix calibration method, and an evaluation method for test kits, belonging to the field of in vitro diagnostics and biometabolic technology. This application establishes a linear correlation between buffer matrix and serum matrix PSA detection results, deriving a specific matrix calibration conversion function that can convert buffer matrix PSA detection values ​​into equivalent serum matrix detection values, effectively offsetting detection errors caused by matrix effects. Experimental data show that after correction using this method, the average absolute bias of PSA detection decreased from 125.82% to 21.01%, and the accuracy after correction meets clinical application requirements. This method enables the replacement of traditional serum matrix calibrators with highly stable, low-cost buffer matrix calibrators, significantly reducing the production and application costs of PSA test kits.

Description

Technical Field

[0001] This invention belongs to the field of in vitro diagnostics and biometrics, and particularly relates to a method for stabilizing prostate-specific antigen standards, a matrix calibration method, and an evaluation method for detection kits. Background Technology

[0002] In the field of in vitro diagnostics, prostate-specific antigen (PSA) is an important serological biomarker for prostate diseases, especially prostate cancer screening, auxiliary diagnosis, and efficacy monitoring. With the development of immunoassay technology, various PSA test kits based on different detection principles have been developed and are widely used in clinical testing.

[0003] However, in practical applications, PSA test kits from different manufacturers, using different testing systems, and even from different batches of the same manufacturer often produce significantly different results. Related research and practical applications indicate that, under the same sample conditions, PSA measurement results obtained from different test kits may exhibit substantial bias, sometimes reaching several times the original value, thus affecting the interpretation of clinical results.

[0004] Currently, each test kit typically relies on its own calibrators or internal calibration systems to assign values. Due to differences in the source, assignment methods, and traceability systems of various calibrators, there is a lack of a unified and comparable numerical basis between different detection systems. Furthermore, existing PSA standard materials often require multiple dilutions to cover the measurement range of the test kit. However, under standard buffer conditions, PSA proteins are prone to adsorption, aggregation, or activity attenuation, thus affecting their numerical stability and the reliability of the test results.

[0005] On the other hand, the actual target of the test kits is human serum samples, while the standards used in some evaluation or calibration processes are usually based on buffer systems, whose physicochemical properties differ from human serum. These matrix differences may introduce systematic bias, making it difficult for evaluation results based on non-serum matrices to accurately reflect the test kit's performance in clinical samples. Therefore, how to reasonably evaluate the performance of PSA test kits while ensuring metrological traceability consistency remains a pressing issue in the field of in vitro diagnostics. Summary of the Invention

[0006] To achieve the above objectives, the present invention adopts the following technical solution: Technical Solution 1: Stabilization System for Traceable PSA Standards To address the insufficient stability of existing PSA reference materials during dilution, storage, and use, this invention proposes a stabilization system based on traceable PSA reference materials. This system, by comprehensively regulating the environment in which the PSA reference materials are processed, reduces the risk of adsorption, aggregation, and activity decay of PSA proteins during handling, without altering their immunological properties and detection response characteristics. This improves their stability and reproducibility in the performance evaluation of in vitro diagnostic kits.

[0007] The stabilization system is used to dilute and utilize traceable PSA reference materials. Its composition and physicochemical properties are optimized to ensure stable detection signal output within the target usage window, exhibiting good intra-batch and inter-batch consistency. PSA reference materials treated with this stabilization system can serve as a reliable quantitative basis for subsequent preparation of working standards, quality control materials, and performance evaluation of diagnostic kits.

[0008] By establishing the aforementioned stabilization system for PSA standards, it becomes possible to evaluate the performance of different test kits based on the same traceable quantitative basis, thus providing a prerequisite for subsequent matrix correlation analysis and kit performance evaluation methods.

[0009] Technical Solution 2: Composition and Preparation Method of Stable Diluent for Traceable PSA Standards Based on the stabilization treatment system described in technical solution 1, this invention further proposes a stable diluent for traceable PSA standards, used for diluting, using, and short-term storing PSA standard substances to improve their quantitative stability and detection consistency in in vitro diagnostic testing processes.

[0010] The stable diluent comprises the following components: Buffer systems, protein protectants, surfactants, salt ion components, and carbohydrate protectants.

[0011] Among them, the buffer system is used to maintain the acid-base stability of the system; the protein protectant is used to reduce the conformational changes and non-specific adsorption of PSA protein during dilution and storage; the surfactant is used to reduce the adsorption loss of PSA protein on the container surface and interface; the salt ion component is used to adjust the ionic strength so that the physicochemical environment of the system is close to the serum conditions; and the carbohydrate protectant is used to improve the stability of the protein during dilution and use.

[0012] By synergistically configuring the above-mentioned components, the resulting stable diluent can effectively inhibit the activity decay of PSA standards during dilution, storage, and detection without affecting the immunological reactivity of PSA, thus ensuring the stability and reproducibility of the detection signal. PSA standards treated with this stable diluent are suitable as a base material for the subsequent preparation of working standards or quality control materials, and can be used to evaluate the performance of different PSA detection kits.

[0013] Technical Solution 3: Preparation Method of PSA Working Standard Based on Stable Diluent Based on the stable diluent described in technical solution 2, this invention further proposes a method for preparing working standards based on traceable PSA reference materials, which is used to construct a series of concentration standard samples covering the measurement range of PSA detection kits to meet the needs of kit performance evaluation and related analysis.

[0014] The preparation method uses traceable PSA standard materials as the source of measurement values, and employs the stable diluent to perform gradient dilutions to prepare PSA working standards at multiple concentration levels. The concentration levels are set according to the measurement range of the PSA detection kit to be evaluated, covering low, medium, and high value ranges to ensure a comprehensive evaluation of the kit's linear range, dynamic response characteristics, and measurement performance.

[0015] During the preparation process, by controlling the dilution method and operating conditions, the working standards at each concentration level maintain good homogeneity and stability, and reduce the value deviations introduced by container adsorption or the operating process. The resulting PSA working standards have a clear source of values ​​and good repeatability, making them suitable for repeated use in different detection systems.

[0016] The PSA working standard prepared by the above method can serve as the basis for subsequent performance evaluation of PSA detection kits and analysis of detection results under different matrix conditions, thereby providing a unified and traceable quantitative basis for performance comparison of different detection systems.

[0017] Technical Solution 4: Correlation and Calibration Method of PSA Detection Results under Different Matrix Conditions To address the practical problem of accurately matching the theoretical concentration levels of PSA standard substances in serum and buffer solutions—two different matrices—while matrix effect correction is still necessary, this invention provides a universal modeling method based on metrological traceability consistency and regression analysis. This method is applicable to PSA test kits with different detection principles, uniformly using the concentration measurements output by the kit as the modeling object. By establishing quantitative relationship models of "theoretical concentration - measured concentration" for both matrices and synthesizing functions within a common range, a matrix calibration function is obtained that can convert buffer solution matrix measurements into equivalent serum matrix measurements. This reduces systematic bias caused by matrix effects and can be used for kit performance evaluation or calibration assignment.

[0018] PSA pure national standard reference material (preferred GBW09334) is prepared separately into two series of standards: Serum matrix calibrators: using human mixed negative serum confirmed to contain no or low levels of endogenous PSA as the matrix, a series of standards are precisely prepared, with their theoretical concentrations denoted as... (Where i = 1, 2, …, n), this series of concentrations covers the measurement range of the target kit. Using the stable diluent described in claim 1 as the matrix, another series of standards are precisely prepared, with their theoretical concentrations denoted as… (Where i=1, 2, …, n) Its concentration range generally overlaps with the serum matrix series (covering the same range), but it is not required that each concentration point be identical or pairable. The theoretical concentrations described are all obtained based on the certified values ​​of national standard substances, precise weighing / volume determination records, and dilution calculations, ensuring that both series of values ​​are traceable to the same national metrological standard.

[0019] Parallel assays and data acquisition: Using the target PSA assay kit to be evaluated or calibrated and its accompanying instruments, two series of standards were measured separately under the same detection conditions to obtain calibrators for the serum matrix. Measurement concentration and buffer matrix standards Measurement concentration Each point can be repeatedly measured and the average value taken to reduce random error.

[0020] Regression models were established for the two sets of data respectively: based on the nominal theoretical concentration of serum matrix standards. As the independent variable, to measure concentration Let be the dependent variable, and the functional relationship be... ,Establish: Preferably, linear regression or piecewise linear regression can be used, let Let be the slope of the linear regression equation. This is the intercept of the linear regression equation; when using linear regression, it can be written as: The nominal theoretical concentration of the buffer matrix standard As the independent variable, to measure concentration Let be the dependent variable, and the functional relationship be... ,Establish: Similarly, linear regression or piecewise linear regression is preferred, let Let be the slope of the linear regression equation. This is the intercept of the linear regression equation; when using linear regression, it can be written as: Where C represents the traceable nominal theoretical concentration variable. The model is used to quantify the difference in the response of a target detection system to the same traceable quantity under two matrices.

[0021] Define a matrix calibration function within the concentration range covered by both series. Used to measure the concentration of any buffer matrix. Converted to serum matrix equivalent measurement concentration : When models S and B are in linear form, the above function can be written as an explicit expression: When the model adopts a piecewise linear form, the corresponding regression parameters are obtained in each concentration segment, and the calibration function is constructed segment by segment in the manner described above.

[0022] The matrix calibration function is used to correct the buffer matrix measurement results to obtain the equivalent measurement concentration of serum matrix, which is used for: kit performance evaluation and kit calibration or result correction assignment, and to improve the consistency and comparability of results between different detection principles / different systems.

[0023] Technical Solution 5: Performance Evaluation Method for Free PSA Detection Kits Based on Traceable Standards This invention further provides a performance evaluation method applicable to PSA test kits with different detection principles. This method uses the same PSA national standard reference material (GBW09334) as the metrological basis, and the working standard / quality control of the buffer matrix prepared with the stable dilution solution of this invention as the main evaluation carrier to obtain repeatable and reproducible evaluation data. Simultaneously, a small amount of serum matrix calibrator is used to establish or verify the matrix calibration function, converting the buffer matrix measurement results into equivalent serum matrix results. This balances standardization and clinical relevance, achieving an objective evaluation of the linearity and accuracy of different test kits, and can output parameters for calibration / correction.

[0024] Prepare a series of working standards / quality control products for the buffer matrix covering the measurement range according to technical solution 2; establish and obtain the matrix calibration function corresponding to the kit according to technical solution 4, or directly call the established calibration function.

[0025] The buffer matrix standards / controls were measured under specified conditions using the kit to be evaluated to obtain the original measured concentrations at each concentration level. The theoretical concentration of the buffer matrix standard was prepared. The independent variable is the original concentration measured by the reagent kit. As the dependent variable, linear or piecewise linear fitting is performed within the target range to obtain the slope, intercept, goodness of fit, and residual distribution, which are used to evaluate the reagent kit's analytical linearity, dynamic range, and risk of high-concentration anomalies (such as the hook effect).

[0026] Used to measure the concentration of any buffer matrix Converted to serum matrix equivalent measurement concentration The theoretical concentration of the buffer matrix standard was prepared. As a reference value, calculate the recovery rate. for: Calculation bias for: The accuracy of the kit can be evaluated based on the recovery rate / bias distribution at each concentration point or in the low / medium / high range. The change in bias before and after correction can be compared to quantify the matrix correction effect.

[0027] Select low / medium / high levels of buffer matrix quality control samples, perform repeated measurements, and calculate CV% to evaluate repeatability; extend to inter-day / inter-batch intermediate precision if necessary. Set several buffer matrix levels in the low concentration range and perform repeated measurements to evaluate the repeatability and detectability of the low value range; if necessary, combine matrix correction functions to provide serum equivalent low value range performance.

[0028] Output the kit's linearity, accuracy (recovery / bias), and (optional) precision and low-concentration performance conclusions; and output / record the corresponding matrix calibration function for subsequent calibration assignment or result correction.

[0029] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses the same traceable prostate-specific antigen standard material as the source of measurement value, which avoids the problem of measurement value separation caused by different test kits relying on their own calibration systems. This makes it possible to evaluate the performance of different test systems on the same measurement value, thereby improving the objectivity of the test kit evaluation results and the comparability between systems.

[0030] (2) In view of the problem that PSA standard substances are prone to adsorption, aggregation or activity decay during dilution, storage and use in the prior art, the present invention introduces the stabilization treatment of standard substances as a pre-step in the performance evaluation method, which effectively improves the stability and repeatability of the standard samples used for evaluation during use, and provides a reliable guarantee for the accuracy of subsequent evaluation results.

[0031] (3) In the evaluation process, the present invention introduces both serum matrix and non-serum matrix standard samples and correlates and processes the detection results under different matrix conditions, so that the standardized evaluation results obtained based on non-serum matrix can reasonably reflect the detection performance under serum sample conditions, thereby solving the problem that it is difficult to balance standardized evaluation and clinical application in the prior art.

[0032] (4) The evaluation method of the present invention does not rely on calibrators or calibration systems provided by the manufacturers of the test kits to be evaluated. Under the premise of maintaining the consistency of traceability of measurement values, PSA test kits from different manufacturers and with different detection principles can be evaluated independently, which is conducive to revealing the performance differences between different detection systems and improving the fairness of the evaluation results.

[0033] (5) This invention organically combines the stabilization treatment of standard substances, the preparation of working standards, the correlation processing of detection results under different matrix conditions, and performance evaluation, forming a complete and operable evaluation method system. It has good repeatability and promotion and application value, and is suitable for performance comparison, methodological verification and related research of PSA detection kits. Attached Figure Description

[0034] Figure 1 This is a comparison curve of the relative luminescence value (RLU) of the PSA standard substance diluted with the diluent of the present invention and the conventional diluent (PBS buffer) in Example 2, respectively, over a period of 24 hours.

[0035] Figure 2This is the overall flowchart of the PSA detection kit evaluation method in Example 4.

[0036] Figure 3 This is a schematic diagram showing the results of evaluating the linear range of a chemiluminescent immunoassay kit using a stable standard substance in Example 4. Detailed Implementation

[0037] Example 1 This embodiment provides a stable diluent screening process: evaluating the impact of different diluent compositions (including Tween concentration, BSA concentration, PBS concentration, NaCl concentration, pH value, and mannitol content) on the stability of a PSA standard (final concentration 7.5 ng / mL). Six key variables were selected, with three levels for each variable. Each PSA working solution was aliquoted into 1.5 mL centrifuge tubes (1 mL per tube, at least 20 tubes) and placed in an incubator at 25°C (simulating room temperature storage conditions). Samples were taken at time points 0, 2, 4, 8, 12, and 24 hours (3 tubes at each time point for repeated measurements, one measurement per sample). Using chemiluminescence immunoassay, each sample was divided into three subsamples, measured once, and the average concentration was calculated. The absorbance value reported by the sample was recorded as the activity value.

[0038] Table 1. Experimental formulations for screening.

[0039] Table 2 Results of Activity Retention Rate Test

[0040] The stable diluent was obtained through the above data screening: 0.01 M PBS (pH 7.4), BSA 0.5% (w / v), Tween-20 0.1% (w / v), NaCl 1.5% (w / v), and mannitol 0.5% (w / v), all of which are g / 100ml.

[0041] Example 2 This embodiment provides the preparation of a stable diluent and the verification of the room temperature stability of PSA standard material: (1) Preparation of stable diluent Prepare a stable dilution solution with the following composition: 0.01 M PBS (pH 7.4), BSA 0.5% (w / v), Tween-20 0.1% (w / v), NaCl 1.5% (w / v), and mannitol 0.5% (w / v). First, dissolve NaCl, BSA, and mannitol sequentially in PBS, mix thoroughly, then add Tween-20 and mix well. If necessary, filter through a 0.22 μm filter membrane for sterilization. Aliquot into low-protein adsorption containers and store at 2–8°C for later use.

[0042] (2) Comparison of diluents 0.01 M PBS (pH 7.4) was used as the control dilution.

[0043] (3) Dilution and storage of PSA standard material Take GBW09334 (converted according to the nominal concentration / content in the certificate), dilute it to the same target concentration with the stable diluent of this invention and the control PBS, mix well and place it at 25°C, and take samples for testing at 0 h, 6 h, 12 h and 24 h respectively.

[0044] (4) Detection and evaluation indicators The signal (e.g., RLU) or output concentration at each time point was measured using a targeted PSA immunoassay system, with the concentration at 0 h recorded as... 24 hours is recorded as Therefore, the immune activity retention rate was calculated with 0 h as the baseline. for: The CV% of repeated measurements at each time point was calculated. The results showed that, compared to PBS dilution, the stable dilution of this invention retained ≥99% of its immunogenicity after 24 h at 25°C, and the CV% was <2%. The stability comparison curve is shown below. Figure 1 As shown.

[0045] Example 3 This embodiment provides the preparation of the evaluation sample system (buffer matrix standard and serum matrix calibrator): (1) Acquisition and pretreatment of serum matrix Healthy donor serum is selected, preferably negative serum that has been screened and confirmed to have undetectable or low background levels of prostate-specific antigen (PSA). To reduce matrix fluctuations introduced by individual differences, it is preferable to mix at least two donor serum samples to prepare a mixed negative serum as the matrix serum. The matrix serum should be free from significant interfering factors such as hemolysis, lipemia, or jaundice; if necessary, low-temperature centrifugation can be used to remove suspended particles.

[0046] (2) Preparation of serum matrix calibrator series Using pure PSA national standard material (preferably GBW09334) as the source of measurement, the spiking solution is prepared by weighing and / or volumetric method, and added to the mixed negative serum in step (1) to obtain a series of serum matrix calibrators. The theoretical concentration of each calibrator is recorded. The concentration points cover the measurement range of the target kit, preferably 5 to 8 levels (e.g., 0.5, 2.0, 10, 50, 100 ng / mL), but it is not required that the concentration points be exactly the same as those of the buffer matrix standard. After each level is spiked, the solution is thoroughly mixed and aliquoted into low protein adsorption containers for storage. To reduce volume / concentration shifts caused by matrix changes, a small volume of high-concentration stock solution can be used for spiking, keeping the spiking volume ratio within a preset threshold (e.g., ≤5% or ≤10%), and the same batch of stock solution is used to complete the entire series of spiking.

[0047] (3) Preparation of a series of working standards for buffer matrix Prepare a stable diluent according to the formulation described in Example 1. Using GBW09334 as the source of measurement, perform serial dilutions in the stable diluent using a weighing method and / or a volumetric method to obtain a series of working standards for the buffer matrix. Record their nominal theoretical concentrations. The concentration range of these standards should roughly overlap with and cover the measurement range of the serum matrix calibrators. If necessary, set up three or more levels of buffer matrix quality control materials (low / medium / high) for precision and routine quality control.

[0048] (4) Dispensing and storage Aliquot the serum matrix calibrators and buffer matrix standards separately into low-protein adsorption centrifuge tubes, labeling them with concentration, batch number, matrix type, and preparation date. Use within 24 hours.

[0049] Example 4 This embodiment provides the establishment of a matrix calibration function and its application in kit accuracy evaluation: The raw data obtained by detecting serum matrix calibrators and stable diluents of PSA at different concentrations using chemiluminescent immunoassay are shown in Table 3.

[0050] (1) Original measurement data Table 3 Theoretical and measured concentrations under two substrates

[0051] (2) Model parameters (regression results) According to the model form of technical solution 4: Model S (serum matrix): The regression results obtained in this embodiment are: , , Model B (buffer matrix): The regression results obtained in this embodiment are: , , (3) Matrix calibration function corresponding to this kit According to the general expression given in technical solution 4: Substituting the parameters from this embodiment, we obtain the specific calibration function for this reagent kit: in The original concentration measured for the buffer matrix sample. This is a measurement of the equivalent concentration of serum matrix.

[0052] (4) Results of bias before and after correction (example of accuracy evaluation) Using the nominal theoretical concentration of the buffer matrix standard as a reference, calculate the uncorrected bias and the postcorrected bias: Table 4 Comparison of bias before and after correction

[0053] The results show that the bias was significantly reduced after correction; based on the data in this embodiment, the average absolute bias decreased from approximately 125.82% to approximately 21.01%. When the residual bias is large in the low concentration range, it is preferable to increase the number of sampling points in the low concentration range or to use a piecewise linear model to further improve the correction accuracy.

[0054] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A diluent for stabilizing prostate-specific antigen standards, characterized in that, It includes the following components at the following concentrations: 0.01M phosphate buffer, pH 7.2–7.6; 0.3%–0.7% bovine serum albumin (BSA); 0.08%–0.12% Tween-20 (TWA); 1.3%–1.7% sodium chloride (SCH); and 0.3%–0.7% mannitol (SCH).

2. The diluent according to claim 1, characterized in that, The pH value of the phosphate buffer solution is 7.

4.

3. The diluent according to claim 2, characterized in that, The bovine serum albumin has a mass-volume percentage of 0.5%.

4. The diluent according to claim 3, characterized in that, The Tween-20 has a mass-volume percentage of 0.1%.

5. The diluent according to claim 4, characterized in that, The sodium chloride has a mass-volume percentage of 1.5%.

6. The diluent according to claim 5, characterized in that, The mannitol has a mass-volume percentage of 0.5%.

7. A matrix calibration method for a prostate-specific antigen detection kit, characterized in that, Includes the following steps: (1) Preparation of standard products: Calibration is performed using PSA certified reference materials of the same traceability level, and two sets of standard products with overlapping concentration ranges are prepared respectively: one set is a gradient series of serum matrix standard products, and the other set is a gradient series of buffer matrix standard products prepared using the diluents described in any one of claims 1 to 6; the gradient series of serum matrix standard products and the gradient series of buffer matrix standard products each contain at least 5 gradient concentration points with a concentration range of 0.5 ng / mL to 100 ng / mL; (2) Parallel detection: The PSA test kit to be calibrated was used to repeatedly test each concentration point of the two sets of standard gradient series, and the average detection value of each concentration point in the serum matrix and the average detection value in the buffer matrix were calculated respectively. (3) Regression fitting: The theoretical concentrations of the two sets of standards were used as independent variables, and the average detection values ​​under the corresponding matrix were used as dependent variables to fit the regression models of serum matrix concentration-detection value and buffer matrix concentration-detection value. (4) Establishment of calibration function: The two regression models are combined and transformed to obtain a matrix calibration function that can convert any PSA detection value under the buffer matrix into an equivalent serum matrix detection value.

8. The matrix calibration method according to claim 7, characterized in that, The regression in step (3) adopts linear regression or piecewise linear regression.

9. A performance evaluation method for a prostate-specific antigen detection kit, characterized in that, Includes the following steps: (1) Using the matrix calibration function pre-established by the matrix calibration method described in claim 7, the original detection values ​​of each concentration point are converted into equivalent serum matrix concentrations; (2) Performance evaluation: Compare the equivalent serum matrix concentration after calibration and conversion with the nominal theoretical concentration of the corresponding serum matrix standard, calculate at least one of the performance indicators of the PSA detection kit to be evaluated, namely accuracy, precision, and linear range, and complete the performance evaluation of the kit.

10. The performance evaluation method according to claim 9, characterized in that, The prostate-specific antigen detection kit is a chemiluminescent immunoassay kit.

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